(1) Field of the Invention
The present invention generally relates to a method and apparatus for identifying large and complex molecules. More particularly, the present invention relates to a method and apparatus for enhanced sequencing of large and complex molecules, including peptides and proteins, using fragment data generated using surface-induced dissociation in conjunction with mass spectrometric analysis.
(2) Description of Related Art
The characterization of large and complex molecules, including biomolecules such as proteins and peptides, has become a focus of applied research in recent years in efforts to advance the field of proteomics. Tandem Mass Spectrometry (MS/MS) is often employed in this effort given its ability to provide backbone structural information through fragmentation of ionized molecules in the gas phase. Fourier Transform Ion Cyclotron Resonance (FT-ICR) Mass Spectrometry (MS) is characterized by high resolution, mass accuracy, and is ideally suited for MS/MS experiments. In typical MS/MS experiments, the ion of interest is mass selected in a first MS step, activated by collision or photon excitation, and the subsequent decay into fragment ions is analyzed in a second MS step. For small ions, a single energetic collision with a neutral gas phase atom is sufficient to dissociate or fragment the ion of interest. Although structural characterization of small molecules is fairly well-established, unambiguous identification of large and complex molecules is limited and often not possible due to poor fragmentation patterns observed in even the best ion activation instruments. Poor fragmentation results in insufficient structure-specific data necessary to characterize the backbone structure of a molecule. Two fundamental limitations constrain the fragmentation of large and complex molecules in MS experiments. First, center-of-mass collision energy decreases with increasing mass of the parent ion, meaning that collision energy provided by collision becomes insufficient to cause fragmentation of a large-mass molecule. Secondly, the density of states within a molecule increases with increasing mass. Thus, with increasing size of a molecule, excitation energy is efficiently redistributed among the numerous vibrational states of the molecule thereby decreasing the fragmentation rate by many orders of magnitude at a given internal energy. It follows that efficient fragmentation of such molecules requires deposition of a large amount of energy into the internal modes of the molecule.
A variety of techniques have been introduced in the art in an attempt to increase the transfer of internal energy deposited to a molecule thereby improving fragmentation, including Multiple Collision Activation-Collision Induced Dissociation (MCA-CID), Sustained Off-Resonance Irradiation-CID (SORI-CID), Infra-Red Multi-Photon Dissociation (IR-MPD), and Surface-induced Dissociation (SID). In MCA-CID, multiple collisions between parent ions of interest and neutral gas atoms such as argon induce fragmentation whereby the ions undergo unimolecular decay yielding fragment ions containing inherent structural information representative of the parent ion. Initially, MCA-CID in FT-ICR mass spectrometry has been achieved using on-resonance excitation whereby the ions are accelerated using an on-resonance radio-frequency (RF) pulse of known amplitude and duration followed by collisional activation with a carrier gas. Unfortunately, on-resonance CID is a poor technique for characterizing large and complex molecules because ions lose kinetic energy in each collision. Thus, multiple-collision activation is inefficient.
To overcome the drawbacks of on-resonance CID for identifying large molecules in FT-ICR MS, different MCA-CID techniques have been employed in the art. For example, Boering et al. report a technique known as Very Low Energy Collision Activation (VLE-CID) in which multiple collisions are achieved using a 180-degree phase shift of the excitation waveform inducing repetitive acceleration and deceleration of ions in the ICR cell to obtain sufficient activation. Lee et al. report a Multiple-Excitation Collision Activation (MECA) technique in which precursor ions not dissociating in a first excitation step are re-excited several times until dissociation occurs. However, implementation of these techniques is rather difficult and has not found widespread application in FT-ICR mass spectrometry. Sustained Off-Resonance Irradiation-CID (SORI-CID) is a widely used MCA-CID technique in which ions under investigation are excited by a radio-frequency (RF) pulse slightly above or below the resonant frequency of the precursor ion thereby causing the ion's kinetic energy to oscillate with time. To ensure multiple collisions, the excitation pulse is applied for a time much longer than the time between collisions such that sufficient energy is accumulated in the internal modes of the ion resulting in fragmentation. Although SORI-CID is widely used for sequencing of large molecules it is well established that it preferentially explores low-energy dissociation channels meaning SORI-CID provides enough structural information only for molecules that readily fragment by many competing low-energy dissociation pathways. However, SORI-CID provides insufficient sequence information for molecules that undergo very specific fragmentation or require very high energies for dissociation. Further, successful application of MCA-CID in FT-ICR MS requires the collision gas to be removed (e.g., a collision gas pump-down delay) prior to mass analysis. If the collision gas is not removed, poor signal and mass resolution result. Low pressures in the ion cyclotron resonance (ICR) cell on the order of 1×10−9 torr are required, necessitating a delay of from 3 to 5 seconds on average to pump out the gas prior to acquisition of MS/MS spectra. Thus, conventional CID and MCA-CID in FT-ICR MS are intrinsically slow analysis techniques.
Infra-Red Multi-Photon Dissociation (IR-MPD) is an alternative method for tandem mass spectrometry. Compared to both on-resonance and off-resonance irradiation, IR-MPD has the advantage that it does not require use of a collision gas. However, because IR-MPD is a very slow activation technique, it has similar disadvantages to SORI-CID. Namely, it follows only the lowest-energy pathways of an ion. In addition, because the fragment ions remain on the axis of the ICR cell during the laser irradiation, they may undergo subsequent fragmentation. To avoid the excessive fragmentation of sequence-informative fragments the duration of the laser pulse is decreased thereby decreasing the overall dissociation efficiency of the precursor ion.
Surface Induced Dissociation (SID) is a technique whereby fragmentation is induced by a single collision of molecules of interest with a surface. SID provides fragmentation at relatively low collision energies (<100 eV). In addition, acquisition of SID spectra in FT-ICR MS does not require introduction of a collision gas into the ICR. cell for ion activation nor the requirement to remove it prior to mass analysis, thus dramatically shortening the acquisition times. Yet, despite the many advancements made by SID, problems are well known in the art. For example, Chorush et al. reported that SID could be used for analyzing large peptides and proteins in FT-ICR MS, but their work demonstrated poorly defined collision energies, incidence angles, collection efficiencies for fragment ions, and low-quality MS/MS. spectra. Introduction of a pulsed gas was further required to confine the fragment ions to the center of the ICR cell prior to detection, making the acquisition time comparable to, or even longer than conventional SORI-CID.
The quantity of ions scattered off an SID surface has been reported to be improved using coated surfaces. Cooks et al. reported use of thin films of self-assembled monolayers (SAMs) of thiols on gold and particularly fluorinated SAMs (e.g., FSAMs). Dongre et al. reported use of thin films of hydrocarbon SAMs (e.g., HSAMs) comprising thiols on gold or silver. Other choices for thin films commonly used in the field include poly-ethers, reported by Koppers et al., Langmuir-Blodgett films on aluminum as reported by Gu et al., and pyrolytic graphite films as reported by Beck et al. Despite the advances made with use of coated surfaces, durability limitations, e.g., temperature durability, are well known in the art and continue to be a concern. Thus, there remains a need for an improved surface for performing SID, particularly for large and/or complex molecules of interest.
An important variable in MS/MS experiments is the time that molecules spend in their activated or excited state prior to detection. Some molecules may have enough energy to fragment but not enough time for dissociation to occur in a particular instrument. Conventionally SID was implemented on double-quadrupole or time-of-flight (TOF) instruments, where the observation time is on the order of 10-100 μs. Typical SID spectra for peptide ions obtained on such instruments contain the primary ion with numerous low-mass fragments. The predominant production of low-mass fragments rarely used for identification of large molecules has resulted in SID spectra for large or complex molecules being largely discounted.
Peptides are biopolymers composed of amino acid residues bonded together via peptide bonds. Peptides and polypeptides are generally asymmetric systems having a beginning NH2 group or N-terminus, and an ending COOH group or C-terminus. Because proteins and peptides are composed of amino acid residues having various side chain “R” groups, in most cases, ions containing such groups are easily and uniquely identified by their measured mass-to-charge (m/z) ratio. Although accurate mass measurement is an important prerequisite for mass spectrometric analyses of large and complex molecules, it is not sufficient for identification. For example, structural isomers have the same m/z in a mass spectrum but different fragmentation patterns upon activation. As a result, structure-specific fragmentation of gas-phase ions is a critical step for peptide and protein sequencing leading to unambiguous identification of the precursor ion or parent molecule. The term “sequencing” as used herein describes any structurally identifying information pertaining to the principal arrangement of monomers in a precursor ion or parent molecule, including fragments thereof. For example, sequencing information includes, but is not limited to, data pertaining to chemical identity, position, and connectivity of the monomers in a molecule of interest. As used herein, identities of residues in a fragment also constitute sequencing information useful in identifying a parent molecule or precursor ion. In contrast, losses of H2O and NH3 from the precursor ion or its subsequent fragments do not contain additional structural information. Designations used herein with reference to specific amino acid residues in a peptide chain follow standard conventions, e.g., alanine (A or Ala), cysteine (C or Cys), aspartic acid (D or Asp), glutamic acid (E or Glu), phenylalanine (F or Phe), glycine (G or Gly), histidine (H or His), isoleucine (I or lle), lysine (K or Lys), leucine (L or Leu), methionine (M or Met), asparagine (N or Asn), proline (P or Pro), glutamine (Q or Gln), arginine (R or Arg), serine (S or Ser), threonine (T or Thr), valine (V or Val), tryptophan (W or Trp), and tyrosine (Y or Tyr).
The general nomenclature for designating backbone fragments resulting from dissociation of peptide ions will now be described. The term “fragment” as used herein refers to any component, material, subcomponent, unit, subunit, segment, section, piece, or portion resulting from the dissociation or fragmentation of an ion or molecule representing less than the complete and intact ion or molecule, e.g., a fragment of a peptide of interest. For example, fragments of a peptide include, but are not limited to, charged species such as bn, an, and yn, generated during dissociation of the peptide, where n denotes the residue position in the intact peptide.
Location of charge along the peptide chain following dissociation designates a fragment as either a b-fragment or y-fragment. For example, b-fragments are formed by cleavage of any peptide bond (i.e., C—N bond between adjacent amino acids) with charge remaining on the N-terminus. By convention, residues in a b-fragment are counted or designated from the left-most residue to the right-most residue. Fragmentation of b-ions results in formation of a-ions. While many potential mechanisms exist for forming a-ions directly from a parent or precursor ion, it is generally accepted that b-ions lose a carbonyl or C=O moiety (28 mass units) to form a-ions, where an=bn−28. Y-fragments are formed by cleavage of any C—N bond between two amino acid residues with charge remaining on the C-terminus. By convention, residues in a y-fragment are counted or designated from the right-most residue to the left-most residue. Other common fragments include ions with masses corresponding to multiple losses of water or losses of NH3, e.g., bn—H2O. Internal fragments formed by cleavage of two backbone bonds are also typical in SID and include both b-type and a-type (b minus 28) fragments. Internal a-type ions composed of only one amino acid are called “immonium” ions.
In general, conventional activation methodologies provide some fragmentation data for large and complex molecules, although in many cases poor fragmentation patterns are obtained using conventional approaches meaning very little new structural information is provided whereby the sequencing may be ascertained and the molecule unambiguously identified. Given the complexity, and ultimate inability to provide sufficient structure-specific fragments to characterize moieties, it is estimated that in excess of 25% of large bio-molecules, including proteins and peptides, remain unidentified in standard MS or tandem MS/MS experiments.
As the current state of the art shows, unambiguous identification of large and complex molecules is complicated by poor dissociation patterns observed in current mass spectrometry instruments. Accordingly, there remains a need to improve structure-specific fragmentation thereby enhancing sequence coverage for identification of large and complex molecules.